The effects of PD-L1 and SCE over-expression on CAR-NK cell cytotoxicity against tumor cells

This experiment aims to investigate how the overexpression of PD-L1 and SCE enhances the cytotoxic activity of CAR-NK cells against tumor cells. Pathway enrichment analysis revealed that CAR-NK cells with reduced HLA-ABC and overexpressed PD-L1 showed enrichment in proliferation and pro-inflammatory pathways. In contrast, CAR-NK cells with reduced HLA-ABC and overexpressed SCE exhibited enrichment in homeostasis-related pathways. On the other hand, cellular stress pathways were more prominently enriched in HLA-ABC reduced CAR NK cells following co-culture with target cells. Overall design: To elucidate the mechanisms underlying the enhanced NK cell cytotoxicity following PD-L1 and SCE overexpression, NK cells isolated from 4 unrelated donors were transduced with lentivectors expressing shRNA #1+MLSN CAR, shRNA #1+MLSN CAR+PD-L1, or shRNA #1+MLSN CAR+SCE. NK cells from the same donors but without transductions were used as controls. NK cells were labeled with CTV and then cocultured with OVCAR8 target cells or in the media alone for 18 hours in triplicates. The cocultured NK cells were purified by cell sorting and processed for RNAseq.